High dietary consumption of fruits and vegetables has been associated with protection against various cancers [1, 2] cardiovascular disease [3], and macular degeneration [4]. Furthermore, it is generally regarded as an important factor for increased energy and overall good health. Due to their widespread distributions in fruits and vegetables, carotenoids can be used as an objective biomarker of fruit and vegetable intake, and carotenoids themselves have been speculated to be one of the anticarcinogenic phytochemicals of plant food [1].
The assessment of carotenoid status has often relied upon the collection of plasma or serum samples for high-performance liquid chromatography (HPLC) analysis. While considered to be the current standard, this approach has several important limitations including high cost, fluctuating carotenoid concentrations in blood (relatively short half-lives), and potential selection bias from participants unwilling to agree to venipuncture.
Assessment of carotenoid status from adipose tissue, a more stable repository for lipid-soluble nutrients like carotenoids, has also been considered in some epidemiological studies. However, this method requires biopsies and more complex sample preparation for HPLC analysis. As a result, a need exists for a highly sensitive, non-invasive, and inexpensive method of carotenoid assessment to objectively evaluate fruit and vegetable intake.
The development of optical monitoring technologies has provided an alternative to HPLC for measurements of carotenoids in human living tissues. In particular, resonance Raman spectroscopy, RRS, has been proposed as an objective indicator of carotenoid status [5, 6]. A novel, non-invasive technique used to measure carotenoid status in the skin using light, RRS utilizes a narrow-wavelength light source in the blue wavelength region to measure total carotenoid concentrations in the skin [7]. The Raman scattered light produces a spectral fingerprint of the carotenoid molecules based on their unique molecular structure and their corresponding unique vibrational energy levels [8].
Because carotenoids from fruits and vegetables accumulate in the dermal layer of the skin, RRS can be used to non-invasively detect the concentration of these molecules. The measurements are based on the resonance Raman response originating from the vibrating carbon backbone common to all carotenoids [5]. More specifically, the backbone's carbon-carbon single bond and double bond stretch frequencies each generate a spectrally sharp Raman signal that is shifted from the excitation light frequency by exactly the amount of the respective vibrational stretch frequency. The intensities of the Raman lines are readily isolated from the excitation light via spectrometer or filter, detected with a linear detector array, and quantified.
One of the preferred body sites for Raman scanning has been the palm of the hand because the dermal melanin pigment is lighter and less variable among individuals of different racial and ethnic backgrounds. Additionally, the stratum corneum, the outer dermal tissue layer is relatively thick in the palm (˜400 μm). This ensures that the excitation light does not penetrate beyond this strongly scattering layer (light penetration depth ˜200 μm) into the deeper tissue layers where it could excite other, potentially confounding chromophores.
RRS used to detect carotenoid levels in the palms of 57 subjects produced a normal distribution [8] with significant width (˜50% of the central value). This implies distinct inter-subject variability, an important characteristic of an objective marker of carotenoid status. It has been shown that carotenoid levels measured with RRS in the inner palm of the hand correlate strongly and significantly with HPLC derived carotenoid levels of fasting serum, thus validating the method in an indirect way [9]. Direct validation experiments have recently been completed that involve skin carotenoid Raman measurements followed by biopsy of the measured tissue volume, and subsequent HPLC analysis [10]. Again, a high correlation was found between both methods.
Reflection spectroscopy has been used previously to measure carotenoid macular pigments in the human retina [11]. Compared to the skin, carotenoid levels in the healthy human macula are about two orders of magnitude higher, and the concentrations of potentially confounding chromophores in the retina are relatively low. Furthermore, the optical media of the human eye that are anterior to the retina are relatively transparent, cause significantly less light scattering, and the sclera of the eye can be used as a light reflector that realizes a more or less straight, double-path, propagation of the excitation light through all tissue layers to the sclera and back. These favorable factors make it possible to use a multi-layer sequential light transmission model, in which the individual absorption and/or scattering effects are described with 8-10 respective absorption and/or scattering coefficients, and in which the macular carotenoid pigment levels are derived from a multi-parameter fit of the calculated reflection spectra to the measured spectra.
In human skin, however, the strong light scattering caused by the outer stratum corneum layer does not permit the assumption of tissue light propagation and modeling of straight light paths. Furthermore, there is no effective internal interface that could be used as a reflector. As a consequence, the methodology of [11] is not applicable. While reflection spectroscopy has been used previously for the measurement of skin carotenoid levels [12, 13], these authors did not provide any details about the data derivation, the presented accuracies were relatively low, and no validation of the method was provided. As a consequence, their approach has not been able to find widespread application.
It is thought that the inhomogeneity of tissue chromophore distributions in living human tissue is a major obstacle in the interpretation of noninvasive reflection spectra [14], and that the diffusion theory of light transport is not valid in turbid media. As a consequence, it is thought that tissue inhomogeneities have to be specifically addressed in measurement schemes that limit the source-detector separation to short distances (in the range of ˜100 μm), and that require complex spectral deconvolution algorithms involving a multi-compartment light propagation model of tissues.
While human skin reflection spectra have been modeled with high accuracy in the spectral absorption range of hemoglobin and oxyhemoglobin absorptions with this approach, the deconvolution of carotenoid absorptions from spectra measured with this approach has been found to be problematic [14] since the signals are “drowned out” or overwhelmed by other confounding chromophore absorptions. The authors of this approach state explicitly that . . . “the analysis of in-vivo spectra regarding beta-carotene is more sophisticated . . . and will be subject to future examination” [14].
A further attempt to derive skin carotenoid concentrations has explored skin color saturation measurements [15]. In this method, color tri-stimulus b-values are measured, and compared to the chromaticity diagram of a white reflection standard. Since the b-value measures the color saturation from the yellow to the blue region, it can be expected to be influenced by the absorption of skin carotenoids occurring in this spectral range. The measurements are influenced, however, not only by the carotenoid absorption, but also by the superimposed absorption and scattering effects of blood and melanin, thus leading to rather unspecific results.
While RRS is potentially a highly molecule specific and highly applicable, field-usable optical skin carotenoid detection method, care has to be taken that the obtained RRS response is adequately interpreted. Different carotenoid species with differing lengths of the conjugated carbon backbone, such as beta carotene on one hand and lycopene on the other, for example, have slightly shifted spectral absorption bands. RRS detection therefore can favor one carotenoid compound over the other if the excitation light overlaps more with one compound than the other.
Since the relative skin concentrations of beta carotene and lycopene are not known a priori, and since they can differ significantly between individuals [8], the RRS responses may not reflect the true composite carotenoid tissue concentrations if this wavelength dependence is not taken into account. Furthermore, RRS detection of skin carotenoids is an absolute detection technique, meaning that the strength of the RRS carotenoid signal response scales linearly with the excitation light intensity and that it can be artificially decreased if unwanted tissue chromophore absorptions and scattering losses exist in the light path. For these reasons care has to be taken to continually calibrate the RRS measurements against an external carotenoid calibration standard, and to limit the RRS measurements to a skin tissue layer that is free of confounding tissue chromophore absorptions. This is best achieved by limiting the excitation and scattered light beam paths to the outermost layer, the stratum corneum, of the palm of the hand. Potential problems may arise if the light propagation in the external carotenoid calibration standard, which is typically an inorganic material, does not adequately simulate the optical properties of the living tissue.
It would therefore be an advance to provide a method and apparatus for an improved safe, noninvasive, rapid, accurate, and specific measurement of the levels of carotenoids and other similar chemical compounds which are present in varying degrees in biological tissues, and to use this information as a diagnostic aid in assessing antioxidant status and detecting malignancy diseases or risk thereof. Specifically, a method is desirable that is less sensitive to variations in skin carotenoid composition, and that does not require calibration with an external carotenoid standard.